Immuno-Scanning Electron Microscopy Reveals That Urease Is Associated with the Microvesicles on the Surface of Helicobacter pylori

Abstract

Background: Microvesicles on the surface of H. pylori is suspected to be sites of bacterial toxin release or artifacts of the autolytic processes of H. pylori during the exponential growth. The molecular nature of the microvesicles has not been revealed yet. In this study, we investigated the structure bound to the commercially available monoclonal antibodies to H. pylori to test whether any of them is related to the microvesicles. Materials and Methods: Because microvesicles are seldom observed by the conventional electron microscopy, we adapted the immuno-scanning electron microscopy (Immuno-SEM). H. pylori 26695 cultured on Mueller Hinton agar plate for 2.5 days were spread onto the 0.5% gelain-coated Thermanox coverslips, and cultured for additional 10 minutes. Immunolabelling was done with monoclonal antibodies MAB921 and MAB922 (Chemicon, Inc.) and 15-nm gold-conjugated protein G. After silver enhancement, the immunolabelled bacteria were processed for scanning electron microscopy. Gold particles were observed using Hitachi S-4700 field emission scanning electron microscope. Molecules against which monoclonal antibodies were prepared were identified by an immunoproteomic technique. Results: Gold particles were observed as glistening white particles especially at the microvesicular domain of H. pylori. Immunoproteomic study revealed that 2 monoclonal antibodies were made against urease. Conclusion: These results suggest that microvesicles of H. pylori may be a site of urease release or have unique molecular components that preferentially bound to urease.